Genomic investigation of the emergence of vanD vancomycin-resistant Enterococcus faecium

Vancomycin-resistant Enterococcus (VRE) is an increasingly identified cause of human disease, with most infections resulting from the vanA and vanB genotypes; less is known about other clinically relevant genotypes. Here we report a genomic exploration of a vanD VRE faecium (VREfm), which arose de novo during a single infectious episode. The genomes of the vancomycin-susceptible E. faecium (VSEfm) recipient and resulting VREfm were subjected to long-read sequencing and closed, with whole-genome alignments, cross-mapping and orthologue clustering used to identify genomic variation. Three key differences were identified. (i) The VREfm chromosome gained a 142.6 kb integrative conjugative element (ICE) harbouring the vanD locus. (ii) The native ligase (ddl) was disrupted by an ISEfm1 insertion. (iii) A large 1.74 Mb chromosomal inversion of unknown consequence occurred. Alignment and phylogenetic-based comparisons of the VREfm with a global collection of vanD-harbouring genomes identified strong similarities in the 120–160 kb genomic region surrounding vanD, suggestive of a common mobile element and integration site, irrespective of the diverse taxonomic, geographical and host origins of the isolates. This isolate diversity revealed that this putative ICE (and its source) is globally disseminated and is capable of being acquired by different genera. Although the incidence of vanD VREfm is low, understanding its emergence and potential for spread is crucial for the ongoing efforts to reduce antimicrobial resistance.


INTRODUCTION
The genus Enterococcus comprises human pathogenic bacteria associated with a diverse range of disease manifestations and high mortality when nosocomially acquired [1,2].The emergence of antibiotic resistance is a further concern, with vancomycinresistant Enterococcus (VRE) listed by the World Health Organization as a high-priority target for the development of novel antimicrobials and therapeutic approaches [3].The large majority of human enterococcal disease is caused by Enterococcus faecalis and Enterococcus faecium; vancomycin resistance in these lineages is predominantly represented by the vanA or vanB genotypes [4].There are, however, an increasing number of alternative van genotypes harboured by enterococci and other bacterial species, many linked with clinical disease: vanC, vanD, vanE, vanG, vanL, vanM and vanN [4][5][6].
A few studies have reported on related genomic islands (GIs) associated with the vanD locus [11,16,17]; carried in different genera and divergent lineages of enterococci.The genes present in this region are suggestive of an integrative conjugative element (ICE).However, the region, while able to be circularized, has not been shown to be mobilizable, and no source for the vanD locus or putative ICE has been conclusively identified [9-11, 13-16, 18, 19], although a recent study has demonstrated that the ICE encompasses genes consistent with those from mobile elements recovered in various anaerobic genera belonging to the order Eubacteriales [17].The limited number and geographical separation of reported vanD VRE suggest that these isolates have arisen primarily through de novo acquisition of the vanD locus [16].This is a concern, as there remains a poor understanding of this phenomena and, importantly, what factors promote this horizontal gene transfer.
Here we report a genomic investigation into the emergence of a vanD vancomycin-resistant E. faecium (VREfm) in New Zealand, which arose de novo during a complex patient infection.Reconstruction of the complete genomes of the vancomycin-susceptible E. faecium (VSEfm) recipient and resulting VREfm enabled characterization of the acquired vanD locus and the putative ICE in which it was carried.Comparison to publicly available vanD-harbouring sequences identified strong similarities between the vanD locus, putative ICE and integration site, suggestive of a common mechanism for horizontal transfer of vanD and a globally dispersed source for this ICE.Collectively, this works provides a summative description of our understanding of the dynamics surrounding the de novo development of vanD VRE.

Bacterial isolates
Isolates were recovered from primary clinical samples using standard laboratory procedures.The first isolate was a VSEfm (named AUS2001), recovered from a blood culture alongside a Morganella morganii and Enterobacter cloacae.The second isolate was a phenotypic VREfm (named AUS2002), recovered 68 days later from a hepatic abscess alongside a Pseudomonas aeruginosa and Lactobacillus rhamnosus.The minimum inhibitory concentrations (MICs), determined by the hospital labratory at the time of isolation using Etest, were 1 and ≥256 mg l −1 for vancomycin, and 2 and ≥256 mg l −1 for teicoplanin, for the VSEfm and VREfm, respectively.

Whole-genome sequencing and genome assembly
Both AUS2001 and AUS2002 underwent DNA extraction (Blood and Tissue kit, Qiagen), following the manufacture's recommended protocol.Sequence libraries were prepared with the Nextera XT DNA Preparation kit (Illumina), and whole-genome sequencing was performed on an Illumina MiSeq with 2×300 bp paired-end chemistry.Genomic DNA was additionally sequenced on the PacBio RS-II with P4+C6 chemistry at the Duke Center for Genomic and Computation Biology (https://genome.duke.edu/).Genome reconstruction was performed as described elsewhere [20].Briefly, long-read sequences were assembled using the HGAP3 pipeline (SMRTPortal v2.3.0)[21].Default parameters were used with the following exceptions: (i) seed read length was set to '1 kb' , (ii) minimum read quality was set to '0.80' , (iii) genome size was set to '3 000 000' bp and (iv) the overlapper error rate was set to '0.04' .Following assembly, contigs were circularized with berokka v0.2.1 ( github.com/ tseemann/ berokka), annotated with Prokka v1.13.3 [22] and reorientated to dnaA (chromosome) or repA (plasmid).All contigs were checked manually for assembly errors, as outlined elsewhere [20], and the final sequences were polished in an iterative manner with the short-read Illumina data using snippy v4.3.6 ( github.com/ tseemann/ snippy), until no variants were detected.In silico MLST was performed

Impact Statement
Vancomycin-resistant enterococci are a pathogen of increasing concern, with vanD representing a rare but clinically important VRE genotype.The limited number of reported cases suggests an evolutionary pattern of de novo emergence; the vanD locus thought to be acquired from the patient's own gut microbiome during prolonged and/or complicated antimicrobial therapy.Here we report the de novo emergence of a vanD VREfm, demonstrating through comparative genomic analysis of paired isolates the likely evolutionary steps taken to become VRE.Further, we place this isolate in the context of publicly available vanD-harbouring sequences and provide a current, summative description of the mechanism of vanD VRE development.

Bioinformatic analysis
De novo assemblies of the short-read data were performed using SPAdes v.3.13.0 [24], excluding the pre-assembly error correction ('--only-assembler'), including post-assembly error correction ('--careful') and a minimum coverage threshold of five ('--covcutoff ') to remove potential contaminants.Mapping-based analyses were performed using snippy v4.6.0 ( github.com/ tseemann/ snippy), with a minimum proportion for variant evidence (--minfrac) of 90 %.For cross-mapping of AUS2001 and AUS2002, the complete genomes and short-read de novo assemblies of each were used as a reference to map the short-read datasets against.Mutations identified in both cross-mapped alignments and not self-mapped alignments were enumerated to estimate the core genetic differences between the two isolates, as described elsewhere [25].Whole-genome comparison of both isolates was performed using blast 2.2.26 [26], and visualized with Artemis Comparison Tool v17.0.1 [27].Further, orthologue clustering to identify all shared and variable coding sequences was performed with roary v3.13.0 [28], using a nucleotide threshold of 99 % without splitting paralogues.
The publicly available vanD-harbouring sequences used in this study are listed in the Table S1, available in the online version of this article.For phylogenetic inference, alignment of the vanD protein, vanD locus (defined as the region from vanR to vanX) and the putative integrative conjugative element (defined as the region between the identified 13 bp direct repeats TTCCC[g/a/c] AC[a/g]ATGA) were generated and neighbour-joining phylogenetic trees built using ClustalW2 [29].Trees were visualized in FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).The vanD protein alignment was also used to categorize sequences by vanD subtype, using the previously reported subtype threshold of ≥97 % sequence identity [13].

Comparison of AUS2001 (VSEfm) and AUS2002 (VREfm)
E. faecium AUS2001 (VSEfm) and AUS2002 (VREfm) represent paired isolates recovered from the same patient 2 months apart during a single, extended admission to a healthcare institution in New Zealand.The recovery of AUS2002 overlapped in time with a large nosocomial outbreak of vanB VREfm in the same institution.Subsequently, AUS2002 was referred to the country's central public health laboratory for genotyping.Discordance between the vancomycin phenotype and vanA/B PCR performed instigated further testing of both AUS2001 and AUS2002.This revealed the isolates to be genetically similar by pulse field gel electrophoresis (PFGE) (Fig. S1), and AUS2002 to carry a vanD gene (by PCR).No other vanD VRE had been recovered in the country around this time, leading to the hypothesis of de novo acquisition, with transfer of the vanD locus suspected to have occurred from the patient's own gut microbiome [30], driven by the extensive and complicated antimicrobial therapy given to the patient during their admission.This unusual finding promoted further genomic investigation to understand the development of AUS2002.
In confirmation of the original PCR and PFGE analysis performed in New Zealand, the genomes of AUS2001 (3 096 160 bp; accession OU015345-OU015349) and AUS2002 (3 241 449 bp; accession OU015350-OU015354) were found to be highly similar.Both genomes belonged to multilocus sequence type (ST) 203.Representing a globally disseminated, clade A enterococcal lineage, ST203 is also a major clone of hospital-associated VRE and has previously been identified to harbour vanD [6,11].AUS2001 and AUS2002 maintained the same four plasmids and cross-mapping of the genomes only identified two single-nucleotide variants, one single-nucleotide polymorphism and one single-base insertion (Table S3).Orthologue clustering of coding sequences in both genomes identified more variation.A total of 2892 clusters were identified using a nucleotide threshold of 99 %.Of these, 2718 clusters were conserved in both genomes and 222 were variably present (Tables S4-S8); 118 of the variable clusters represented the mobile element described below.
Whole-genome alignment of AUS2001 and AUS2002 identified three large structural genomic differences, all within the chromosome (Fig. 1); two were considered the most relevant to the change in vancomycin susceptibility.First, AUS2002 contained an insertion of 142 597 bp, the region flanked by 13 bp direct repeats (5' TCATTGTCGGGAA 3') and encoding a putative 171 proteins (Fig. 1, Table S2).This included an integrase (int) gene and the three most conserved components of conjugative elements, a relaxase (mobC), a coupling protein (virD4) and the ubiquitous type IV secretion system (virB4) (Fig. 1c) [31].Further, the region maintained a higher average GC content (44 %) than the rest of the genome (38 %) (Fig. 1b), strongly suggestive of horizontal gene transfer from a nonenterococcal host.Integration occurred directly downstream of the fifth (of six) ribosomal RNA loci disrupting lysS (Fig. 1c), as has been previously described [16].The integrated region also contained the vanD locus -the source of the acquired vancomycin resistance in AUS2002.Based on these observations, this element was deemed a putative ICE that we have termed ICEVREfm_vanD.Of note, the mobility-associated genes identified encoded an incomplete system, notably missing an excisionase.While the ICE may encode an unidentified excision module such as a transposase [32,33], it may also be the case that ICEVREfm_vanD is not fully autonomous and relies on an externally encoded trans-acting protein(s) or utilizes a different mechanism for transfer.The lack of evidence for excision of the ICE is consistent with the vanD VRE literature [10,[13][14][15][17][18][19].
Second, the vanD genotype (excluding subtype vanD2) has invariably been associated with vanRS mutations and/or impairment/ disruption of the native chromosomal ligase, encoded by ddl [9,16,34,35].Examination of the ddl gene in AUS2002 found an ISEfm1 inserted centrally in the coding region of the transposase at nucleotide position 759/1077, consistent with previous reports [12,19].It is predicted that this insertion will prevent production of the normal d-ala-d-ala peptide.While this would augment the efficiency of the resistance mechanism (maximizing inclusion of the new d-ala-d-lac peptide, which is unaffected by vancomycin [18], in the cell wall), it renders AUS2002 dependent on the acquired vanD ligase for cell wall cross-linking.As the sole functional ligase, loss of vanD through excision of the ICE would likely be highly detrimental if not lethal to the cell and may be promoting stabilization and maintenance of the ICE.AUS2002 can be grown both in the presence and absence of vancomycin, suggesting that the system is constitutively expressed in this isolate.Further, four mutations were identified in vanS (Fig. 1c); all (V67A, K308Q, I309V and R335Q) have previously been identified and linked to constitutive expression of the vanD locus [9].Third, was a large chromosomal inversion of 1.74 Mb (Fig. 1a), occurring between two ribosomal RNA loci.It is unclear whether the inversion was prompted by the integration of ICEVREfm_vanD or has occurred independently.However, other large chromosomal inversion events have been reported to have coincided with vanD acquisition [16], suggestive of a potential, possibly transient, state of genomic instability surrounding the integration event.In the case of AUS2002, the region inverted does not overlap with the integration site of the ICE.It is unclear what phenotypic impact this inversion (or those reported in other isolates) has had, but it is an avenue for future investigations when exploring the phenomena of de novo acquisition of vanD.

Comparison of AUS2002 with all available vanD-harbouring genomes
To place AUS2002 in the context of other vanD-harbouring genomes, a search of the vanD literature and the National Center for Biotechnology Information's (NCBI's) sequence databases was completed, with 49 sequence records representing unique isolates identified (Table S1).
Comparison of sequences from all 50 isolates identified that the vanD protein in AUS2002 is most closely related to the vanD4 subtype (Fig. 2), sharing 98 % amino acid identity with all other vanD4 protein sequences.However, AUS2002 also shared 97 % amino acid identity with all vanD5 protein sequences.Historically, the vanD subtypes have been defined with a 97 % amino acid identity threshold.But AUS2002 and other more recently identified vanD proteins have highlighted that this threshold no longer enables classification of vanD proteins into discrete subtypes, with vanD subtypes 1, 2 and 3 forming one phylogenetically congruent group, and subtypes 4 and 5 a second group alongside other related vanD proteins (Fig. 2).Two novel vanD protein subtypes were also identified and form a distinct monophyletic cluster in the vanD protein tree (Fig. 2).A phylogenetic tree constructed from an alignment of the full vanD locus for 48/50 isolates (2 sequence records did not contain the full locus sequence) (Fig. 3a) revealed a topology largely consistent with the tree constructed for the vanD peptide (Fig. 2).Of note, the topology did not show any strong correlation with the geographical location or year of isolation for isolates, or the species the sequence was recovered from.This finding is consistent with the hypothesis that de novo generation is the predominant mechanism by which vanD VRE emerge.
The putative ICE in AUS2002 was found to share significant similarity in sequence and structure to the 30 other full-length mobile elements identified in the vanD harbouring sequences (Fig. 3b).The integration site (in lysS) was conserved in all isolates (n=36 sequences with partial or full-length ICE), irrespective of the taxonomic, geographical and host origins of the isolates (Table S1).A direct repeat sequence of 13 bp (TTCCC[g/a/c]AC[a/g]ATGA) with 11 sites conserved was identified; variations in the 2 variable sites does not correlate with the phylogeny for the ICE (Fig. 3b).The findings from this comparative genomic analysis are consistent with previous smaller comparisons [11,16,17], and collectively provide strong evidence for a common mobile element for the vanD locus.Phylogenetic trees constructed for both the vanD locus (Fig. 3a) and the ICE sequences (Fig. 3b) were highly congruent in their structure, suggesting that both elements are commonly mobilized together.Collectively, these investigations support a hypothesis that the identified ICE is the principal element facilitating mobility of the vanD locus and is likely highly specific in its target site for integration.

CONCLUSIONS
Here we present a case study of the de novo emergence of vanD VREfm during a complex and prolonged infection and add to the understanding of the genomic mechanisms involved.Complete genome reconstruction enabled identification of the vanDharbouring putative ICE.The element does not appear to be autonomously transferable and is stably fixed within the AUS2002 chromosome.This likely represents a stepwise adaptive process in AUS2002, resulting from prolonged antibiotic exposure; acquisition of ICEVREfm_vanD into AUS2001 to gain glycopeptide resistance, followed by disruption of the native ddl ligase to enhance the resistance mechanisms and a large chromosomal inversion of unknown impact (the order of the last two events is unclear).While a resistance phenotype was gained, a consequence is that AUS2002 is likely now dependent on the acquired vanD ligase for cell wall cross-linking and excision of the ICE may be lethal to the cell.Comparison of the limited number of available vanD-harbouring sequences suggested that many vanD VRE have evolved through a similar evolutionary pathway to AUS2002.The diverse taxonomic, geographical and host origins for these isolates largely preclude transmission as an explanation for the spread of the vanD locus, with de novo generation being far more plausible.However, for this to occur the origin species or other carriers of the putative ICE would need to be globally disseminated.In lieu of regular molecular or sequence-based typing, the frequency with which vanD VRE emerge is unknown.Further, the circumstances that promote mobilization of the ICE are still unclear, but have important implications for managing the risk of future vanD VRE emergence events.

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Response to reviewers Reviewer 1 Comments to Author:
We thank Reviewer 1 for their positive remarks on our manuscript and have addressed all comments.Please find our point-by-point responses below.
I found this work to be very thoroughly conducted, presented in a clear and precise manner, with clear figures and text.
My only point of contention is minor -there are some sentences that I feel should contain references to back up the statements made.I have included those statements below, as well as a few other minor comments.Otherwise this is a great piece of research which I recommend for publication.
L67 -Enterococcus represents a genus of human pathogenic bacteria, associated with a diverse range of disease manifestations and high mortality when nosocomially acquired.

We have added two citations to support this statement -Arias & Murray, 2012; Fiore et al., 2019.
L71 -The large majority of human enterococcal disease is caused by E. faecalis and E. faecium; vancomycin resistance in these lineages predominantly represented by the vanA or vanB genotypes.
We have added one citation to support this statement -Ahmed & Baptiste, 2018.
L114-The minimum inhibitory concentrations (MICs), determined by Etest at the time of isolation were 1 mg/L and ≥ 256 mg/L for vancomycin, and 2 mg/L and ≥ 256 mg/L for teicoplanin, for the VSEfm and VREfm respectively.
There is no citation available for this data.These values were determined by the hospital lab during the original identification and characterisation of these isolates -the data made available for this manuscript from co-author SR.We have amended the wording of this statement to clarify this.
Lines 117 -119: "The minimum inhibitory concentrations (MICs), determined by the hospital lab at the time of isolation using Etest, were 1 mg/L and ≥ 256 mg/L for vancomycin, and 2 mg/L and ≥ 256 mg/L for teicoplanin, for the VSEfm and VREfm respectively." L129 -"All contigs were checked manually for assembly errors"; what is the criteria for the manual assessment of assembly errors?
The process employed for assembly error detection is outlined in a previous publication from our group.We have added this citation to the text (at line 133) -Baines et al., 2020.

Reviewer 2 Comments to Author:
We thank Reviewer 2 for their positive remarks and suggestions on additional results to improve the manuscript.All comments have been addressed.Please find our point-by-point responses below.
This is an article comparing 2 Enterococcus faecium genomes, from strains isolated from the same patient, 2 months apart.In the second isolate they find that an ICE sequence has been inserted, which confers resistance to vancomycin.This is relevant and is a remarkable contribution, which helps to understand how antibiotic resistance spreads between species.The methodology is adequate to analyze this sequence and present its expansion.I would just like to see the complete comparison between both isolates: genes present and absent in the second isolate, and total number of variants between both genomes.
To expand on our comparison of the two genomes, we have performed orthologue clustering to enable enumeration of shared and variably present genes.The results of this analysis have been added to the text.
Lines 204-207 (results): Orthologue clustering of coding sequencing in both genomes suggested more variation.A total of 2892 clusters were identified using a nucleotide threshold of 99%.Of these, 2718 clusters conserved in both genomes and 222 were variable present (Table S4-S8); 118 of the variable clusters represented the mobile element described below." All orthologue clustering data has been added to the supplementary materials (Tables S4-S8).Further, the format of the supplementary materials has been changed (from pdf to excel) to make the data more usable.
I also add a number of points to be addressed: 185: Does it refer to the whole genome or only to plasmids?Two variants seems really few.
Yes, there were only 2 confirmed variants identified across both genomes (chromosomes and plasmids) using the cross-mapping approach outlined in the methods.We did observe 80 additional variants (almost exclusively insertion/deletions) that were only observed when the reads of AUS2001 were mapped to the complete AUS2002 genome.We suspected that these were technical errors resulting from the pacbio sequencing (as these genomes were sequenced > 5 years ago when the technology was less accurate).However, we have provided the full list of variants in the supplementary materials (Table S3).
Fig. 1: Which identity threshold has been used?Red means passing that threshold and blue the same but inverted?In any case, the number of variants found throughout the genome should be indicated in the results to better estimate the divergence between the two genomes.
A minimum identity threshold of 90% (nucleotide) was used.For all matches that meet this threshold, they are coloured red if found in the same orientation in both genomes, and blue, if found in opposite orientations.This information has been added to the legend for Figure 1.
Lines 323-324."Only blast matches of ≥90% nucleotide identity are shown; red indicates matches in the same orientation and blue indicates opposite orientations." Further information about the number of variants has been provided (please refer to the response for the previous comment).
Fig. 2: Do the numbers in parentheses represent the year of isolation?
Yes.The legend for figure 3 has been amended to clarify this.
Lines 337-339." Individual isolate information on taxonomy, geographic continent (tip circle colour) and year of recovery (value provided in parentheses of tip annotation), host (the single animal isolate indicated with the mouse symbol), and the ICE direct repeat sequences (tip square colour) are provided in the tip labels and symbols (refer to key)." The fact that ICE appears in different genres should be highlighted more.For example, making a Blast of its sequence, it is found in Clostridia or Blautia, at 96% identity.Is it expected to be the same ICE?
Yes, the taxonomic diversity of this ICE is highly interesting and is already the focus of an existing publication that we have cited.This paper has demonstrated that there is strong conservation of a number of the ICE genes (they term them the ICE 'backbone') across diverse anerobic species.We are not exactly clear what the reviewer is requesting here.If this is in reference to the vanD subtyping scheme (Figure 2), this was established when only a handful of vanD sequences had been identified, using a common threshold of 97% amino acid identity to clusters sequences into allele type.We have included the scheme in our work to (i) enable comparison of new isolates to the historic vanD subtype, but (ii) also demonstrate that with the increasing number of vanD sequences now available, the 97% clustering threshold no longer clearly separates sequences into exclusive groups (Figure 2).However, regardless of subtype, all vanD alleles mediate phenotypic resistance to vancomycin.
Data not shown should be given as Supplementary Figures, to confirm results obtained.
As our findings from the PCR performed to investigate mobility of the ICE was consistent with numerous pervious vanD publications we have opted to remove it from the manuscript.

VERSION 1
Editor Comments: This is an article comparing 2 Enterococcus faecium genomes, from strains isolated from the same patient, 2 months apart.In the second isolate they find that an ICE sequence has been inserted, which confers resistance to vancomycin.This is relevant and is a remarkable contribution, which helps to understand how antibiotic resistance spreads between species.The methodology is adequate to analyze this sequence and present its expansion.I would just like to see the complete comparison between both isolates: genes present and absent in the second isolate, and total number of variants between both genomes.I also add a number of points to be addressed: 185: Does it refer to the whole genome or only to plasmids?Two variants seems really few.Fig. 1: Which identity threshold has been used?Red means passing that threshold and blue the same but inverted?In any case, the number of variants found throughout the genome should be indicated in the results to better estimate the divergence between the two genomes.Fig. Comments: I found this work to be very thoroughly conducted, presented in a clear and precise manner, with clear figures and text.My only point of contention is minor -there are some sentences that I feel should contain references to back up the statements made.I have included those statements below, as well as a few other minor comments.Otherwise this is a great piece of research which I recommend for publication.L67 -Enterococcus represents a genus of human pathogenic bacteria, associated with a diverse range of disease manifestations and high mortality when nosocomially acquired.L71 -The large majority of human enterococcal disease is caused by E. faecalis and E. faecium; vancomycin resistance in these lineages predominantly represented by the vanA or vanB genotypes.L114-The minimum inhibitory concentrations (MICs), determined by Etest at the time of isolation were 1 mg/L and ≥ 256 mg/L for vancomycin, and 2 mg/L and ≥ 256 mg/L for teicoplanin, for the VSEfm and VREfm respectively.L129 -"All contigs were checked manually for assembly errors"; what is the criteria for the manual assessment of assembly errors?L188 -"three large structural genomic difference"; spelling, differenceS

Please rate the quality of the presentation and structure of the manuscript Good
To what extent are the conclusions supported by the data?Strongly support

Fig. 1 .
Fig. 1.Genomic location and structure of the putative integrative conjugative element (ICE) harbouring the vanD locus.(a) Whole-genome alignment of AUS2001 (VSEfm) and AUS2002 (VREfm).Only blast matches of ≥90 % nucleotide identity are shown; red indicates matches in the same orientation and blue indicates opposite orientation.Highlighted are the three major structural changes identified: (1) integration of the putative ICE, (2) disruption of ddl through gain of an ISEfm1 and (3) a large chromosomal inversion.(b) Graph of the GC content across the genome of AUS2002, highlighting the increase in GC% in the ICE region.(c) Schematic of the ICE, highlighting the genes constituting an incomplete conjugation system and the vanD locus.Mutations identified in vanS are annotated and considered to mediate constitutive expression of the vanD locus.

Fig. 2 .
Fig.2.Diversity of the vanD protein sequence.A neighbour-joining phylogenetic tree was generated from an alignment of the vanD protein for all identified vanD-harbouring sequences (n=50) using ClustalW.The pairwise percentage amino acid identity is provided in the heatmap.Clusters have been defined using a 97 % identity threshold; solid lined boxes indicate inclusive groups (i.e.sharing ≥97 % identity with one other member) and dotted line boxes indicate explicit groups (i.e.sharing ≥97 % identity with all members).Scale bar represents number of sites.

Fig. 3 .
Fig. 3. Phylogenetic congruence of the vanD locus and putative integrative conjugative element.Neighbour-joining phylogenetic trees were generated from alignments of the vanD locus (a), n=48 sequences, and the putative integrative conjugative elements (b), n=31 sequences, using ClustalW.Sequences represented in both trees are joined to highlight similarities in topology.Individual isolate information on taxonomy, geographical continent (tip circle colour) and year of recovery (value provided in parentheses of tip annotation), host (the single animal isolate indicated with a mouse symbol) and the direct repeat sequences (tip square colour) are provided in the tip labels and symbols (refer to key).Scale bar represents number of sites.

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Thank you very much for submitting this revised manuscript to Access Microbiology and addressing the reviewer suggestions.I am pleased to inform you that your manuscript is now accepted for publication.Congratulations to all authors!

recommendation and comments https
://doi.org/10.1099/acmi.0.000712.v1.5 © 2023 de Dios R.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Thank you very much for submitting your manuscript to Access Microbiology.It has now been reviewed by two experts in the field, with whom I agree that this is indeed a valuable contribution to understand the spread of antibiotic resistance.Please find below the reviewer comments and suggestions to improve your manuscript.Please pay special attention to those concerning the addition of references, a deeper comparison between the isolates, the addition of information on the vancomycin resistance genotypes and the suggestion to highlight the ICE.With respect to the data not shown, as per the open data policy of Access Microbiology, I will kindly ask you to provide them as supplementary material.
2: Do the numbers in parentheses represent the year of isolation?The fact that ICE appears in different genres should be highlighted more.For example, making a Blast of its sequence, it is found in Clostridia or Blautia, at 96% identity.Is it expected to be the same ICE?I am missing more information on what the vancomycin resistance genotypes are based on.Data not shown should be given as Supplementary Figures, to confirm results obtained.Please rate the manuscript for methodological rigour GoodPlease rate the quality of the presentation and structure of the manuscript Very goodTo what extent are the conclusions supported by the data?Strongly supportDo you have any concerns of possible image manipulation, plagiarism or any other unethical practices?NoIs there a potential financial or other conflict of interest between yourself and the author(s)?NoIf this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes Reviewer 1 recommendation and comments https://doi.org/10.1099/acmi.0.000712.v1.3 © 2023 Barber J.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.